Cell cycle–dependent centrosome clustering precedes proplatelet formation

Platelet-producing megakaryocytes (MKs) primarily reside in the bone marrow, where they duplicate their DNA content with each cell cycle resulting in polyploid cells with an intricate demarcation membrane system. While key elements of the cytoskeletal reorganizations during proplatelet formation have been identified, what initiates the release of platelets into vessel sinusoids remains largely elusive. Using a cell cycle indicator, we observed a unique phenomenon, during which amplified centrosomes in MKs underwent clustering following mitosis, closely followed by proplatelet formation, which exclusively occurred in G1 of interphase. Forced cell cycle arrest in G1 increased proplatelet formation not only in vitro but also in vivo following short-term starvation of mice. We identified that inhibition of the centrosomal protein kinesin family member C1 (KIFC1) impaired clustering and subsequent proplatelet formation, while KIFC1-deficient mice exhibited reduced platelet counts. In summary, we identified KIFC1- and cell cycle–mediated centrosome clustering as an important initiator of proplatelet formation from MKs.


Fig S3
) Cytotoxicity of the inhibitors SR31527 (SR), CW069, griseofulvin (Gris) and CCB02 in hematopoietic stem and progenitor cells (HSPCs) cultured for 3 days (A) as well as mature BMMKs cultured for 24h (B) was assessed using an LDH cytotoxicity assay per the manufacturers' instructions.Absorbance was measured at a plate reader.(A) N=2; (B) N=1; 3 technical replicates.Data are presented as mean ± SD. (C) Simulation-based modeling of interactions between chromosomes and centrosomes [64,65]: Kinesin-14 motors like KIFC1 (red) stretching from two centrosomes generate effective intercentrosomal attraction forces (red arrows) (1).Microtubules interacting with dynein motors (blue) on the kinetochores are pulled toward the chromosomes causing the effective attraction force between the centrosome and chromosome (blue arrow).Microtubules interacting with chromokinesin motors (purple) on the chromosome arms are pushed away from the chromosomes causing the effective repulsion force between the centrosome and chromosome (purple arrow).Two opposing centrosome-chromosome forces position centrosomes at a characteristic distance from the chromosomes (2).The chromosomes aggregate to several clusters at the center, while the effective attraction clusters some of the centrosomes and localizes them around the chromosomes resulting in multipolarity.The centrosomal clusters do not aggregate further because the attraction between them is either blocked by the chromosomes in between (dotted arrows) or is too weak because of the large inter-centrosomal cluster distances (dashed arrows).Following DNA decondensation, the only remaining force is the attraction between the centrosomal clusters (3).This KIFC1-generated attraction force clusters all centrosomes together.The resulting combined microtubule aster is so dense that crosslinking action of MAPs could lead to bundling of microtubules (4), from which they are distributed to the cell cortex.

Fig S4. (A)
PCR for wildtype (WT) and mutant (KO) Kifc1 allele using DNA derived from Kifc1 -/-, heterozygous (Kifc +/-) and WT mice.(B, C) Analysis of litter sizes (B) and sex distribution among the litters (C) of WT and Kifc1 -/-mice.Unpaired, two-tailed student's t-test.P < 0.05.(D, E) Red and white blood cell counts in WT and Kifc1 -/-mice were assessed using an automated blood cell analyzer.N=25.Unpaired, two-tailed student's t-test.(F) Platelet recovery after depletion was assessed using an automated blood cell analyzer.N=3.(G) Immature platelet fraction in WT and Kifc1 -/-mice was assessed using an automated blood cell analyzer.N=25.Unpaired, two-tailed student's t-test.(H, I) Activation of platelet αIIbβ3 integrins and exposure of P-selectin were analyzed by flow cytometry upon stimulation with different agonists.N=4.Unpaired, two-tailed student's t-test.P < 0.05.(J) Percentage of MKs expressing CD41 and CD42d after 4 days of culturing was assessed by flow cytometry.N=3.K) Number of MKs containing clustered centrosomes was quantified by immunofluorescence stainings for pericentrin and α-tubulin.N=4.Unpaired, two-tailed student's t-test.P < 0.001 ***.(L) TPO levels in plasma derived from WT or Kifc1 -/-mice were analyzed using an enzyme-linked immunosorbent assay.N=6.All data are presented as mean ± SD.

Gene Protein
Method Function

Aurka
Aurora Kinase A Polysome Profiling [48] Associates with the centrosome and the spindle microtubules during mitosis and plays a critical role in various mitotic events [90].

STAR Related Lipid Transfer Domain Containing 9
Ubiquitin Pulldown Microtubule-dependent motor protein required for spindle pole assembly during mitosis and to stabilize the pericentriolar material (PCM) [94].

Centrosomal Protein 192 Ubiquitin Pulldown
Required for mitotic centrosome and spindle assembly.Appears to be a major regulator of PCM recruitment and centrosome maturation [95].